FOXF1 Regulates Alveolar Epithelial Morphogenesis through Transcriptional Activation of Mesenchymal WNT5A

Mutations in the FOXF1 (forkhead box F1) gene, encoding the mesenchymal FOX (forkhead box) transcription factor, are linked to alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with the loss of alveolar capillaries and lung hypoplasia. Although proangiogenic functions of FOXF1 have been extensively studied, the role of FOXF1 in mesenchymal-epithelial signaling during lung development remains uncharacterized. Herein, we used murine lung organoids to demonstrate that the S52F FOXF1 mutation (found in patients with ACDMPV) stimulates canonical WNT/β-catenin signaling in type 2 alveolar epithelial cells (AEC2s), leading to increased proliferation of AEC2s and decreased differentiation of AEC2s into type 1 alveolar epithelial cells (AEC1s). Alveolar organoids containing Foxf1^WT/S52F lung fibroblasts and wild-type epithelial cells grew faster on Matrigel and exhibited AEC2 hyperplasia. AEC2 hyperplasia and loss of AEC1s were found in the lungs of Foxf1^WT/S52F embryos, a mouse model of ACDMPV. Activation of canonical WNT/β-catenin signaling in AEC2s of lung organoids and Foxf1^WT/S52F mice was associated with decreased expression of noncanonical WNT5A (Wnt family member 5A) ligand in lung fibroblasts. Mechanistically, FOXF1 directly activates the Wnt5a gene transcription through an evolutionarily conserved +6320/+6326 region located in the first intron of the Wnt5a gene. Site-directed mutagenesis of the +6320/+6326 region prevented the transcriptional activation of the Wnt5a enhancer by FOXF1. Treatment with exogenous WNT5A ligand inhibited the effects of the S52F FOXF1 mutation on canonical WNT/β-catenin signaling in alveolar organoids, preventing aberrant AEC2 expansion and restoring differentiation of AEC1s. Activation of either FOXF1 or WNT5A may provide an attractive strategy to improve lung function in patients with ACDMPV.